T helper type 1 (Th1)-type CD4+ antitumor T cell help appears critical to the induction and maintenance of antitumor cytotoxic T lymphocyte (CTL) responses in vivo. In contrast, Th2- or Th3/Tr-type CD4+ T cell responses may subvert Th1-type cell-mediated immunity, providing a microenvironment conducive to disease progression. We have recently identified helper T cell epitopes derived from the MAGE-6 gene product; a tumor-associated antigen expressed by most melanomas and renal cell carcinomas. In this study, we have assessed whether peripheral blood CD4+ T cells from human histocompatibility leukocyte antigens (HLA)-DRβ1*0401+ patients are Th1- or Th2-biased to MAGE-6 epitopes using interferon (IFN)-γ and interleukin (IL)-5 enzyme-linked immunospot assays, respectively. Strikingly, the vast majority of patients with active disease were highly-skewed toward Th2-type responses against MAGE-6–derived epitopes, regardless of their stage (stage I versus IV) of disease, but retained Th1-type responses against Epstein-Barr virus– or influenza-derived epitopes. In marked contrast, normal donors and cancer patients with no current evidence of disease tended to exhibit either mixed Th1/Th2 or strongly Th1-polarized responses to MAGE-6 peptides, respectively. CD4+ T cell secretion of IL-10 and transforming growth factor (TGF)-β1 against MAGE-6 peptides was not observed, suggesting that specific Th3/Tr-type CD4+ subsets were not common events in these patients. Our data suggest that immunotherapeutic approaches will likely have to overcome or complement systemic Th2-dominated, tumor-reactive CD4+ T cell responses to provide optimal clinical benefit.

In melanoma, the RAS/RAF/MEK/ERK signalling pathway is an area of great interest, because it regulates tumor cell proliferation and survival. A varying mutation rate has been reported for B-RAF and N-RAS, which has been largely attributed to the differential source of tumor DNA analyzed, e.g., fixed tumor tissues or in vitro propagated melanoma cells. Notably, this variation also interfered with interpreting the impact of these mutations on the clinical course of the disease. Consequently, we investigated the mutational profile of B-RAF and N-RAS in biopsies and corresponding cell lines from metastatic tumor lesions of 109 melanoma patients (AJCC stage III/IV), and its respective impact on survival. 97 tissue biopsies and 105 biopsy-derived cell lines were screened for B-RAF and N-RAS mutations by PCR single strand conformation polymorphism and DNA sequencing. Mutations were correlated with patient survival data obtained within a median follow-up time of 31 months. B-RAF mutations were detected in 55% tissues and 51% cell lines, N-RAS mutations in 23% tissues and 25% cell lines, respectively. There was strong concordance between the mutational status of tissues and corresponding cell lines, showing a differing status for B-RAF in only 5% and N-RAS in only 6%, respectively. Patients with tumors carrying mutated B-RAF showed an impaired median survival (8.0 versus 11.8 months, p = 0.055, tissues; 7.1 versus 9.3 months, p = 0.068, cell lines), whereas patients with N-RAS-mutated tumors presented with a favorable prognosis (median survival 12.5 versus 7.9 months, p = 0.084, tissues; 15.4 versus 6.8 months, p = 0.0008, cell lines), each in comparison with wildtype gene status. Multivariate analysis qualified N-RAS (p = 0.006) but not B-RAF mutation status as an independent prognostic factor of overall survival. Our findings demonstrate that B-RAF and N-RAS mutations are well preserved during short term in vitro propagation and, most importantly, differentially impact the outcome of melanoma patients.

Prostaglandin E 2 (PGE 2 ) is an inflammatory mediator often used to increase CCR7 expression in the dendritic cells (DCs) used as cancer vaccines and to enhance their responsiveness to lymph nodeassociated chemokines. Here, we show that high surface expression of CCR7 on PGE 2 -matured DCs is associated with their suppressed production of the endogenous CCR7 ligand, CCL19, and is reversible by exogenous CCL19. In contrast to the PGE 2 -matured DCs, DCs matured in the presence of toll-like receptor (TLR) ligands and interferons produce high levels of both CCL19 and CCR7 mRNA/ protein, but show selectively reduced expression of surface CCR7, which is compensated after DC removal from the CCL19-rich maturation environment. In accordance with these findings, PGE 2 -matured DCs show significantly higher in vitro migratory responsiveness to lymph node-associated chemokines directly after DC generation, but not after additional short-term culture in vitro, nor in vivo in patients injected with 111 indium-labeled DCs. The differences in CCL19-producing ability imprinted during DC maturation result in their different abilities to attract CCR7 ؉ naive T cells. Our data help to explain the impact of PGE 2 on CCR7 expression in maturing DCs and demonstrate a novel mechanism of regulatory activity of PGE 2 , mediated by the inhibition of DCs ability to attract naive T cells. (Blood. 2010;116(9):1454-1459) IntroductionProstaglandin E 2 (PGE 2 ) is an inflammatory mediator with suppressive activity at several levels of the immune response. 1-3 PGE 2 selectively impairs the production of interleukin-2 (IL-2) and interferon-␥ (IFN␥) in T cells, 4,5 inhibits the responsiveness to T cell-activating and Th1-driving cytokines such as IL-2 and IL-12p70, 6,7 blocks the production of dendritic cell (DC)-produced proinflammatory cytokines, including IL-12p70, [8][9][10] and induces the production of IL-12R antagonist IL-12p40 homodimer. 11,12 Recently, PGE 2 has been also shown to promote the ability of DCs to preferentially attract the inhibitory regulatory T cell (Treg) subset of CD4 ϩ T cells 13 and to directly promote the development of Tregs. 14,15 In apparent contrast to these suppressive functions, PGE 2 also has been reported to synergize with tumor necrosis factor-␣ (TNF␣) in the induction of DC maturation 10,16 and in promoting CCR7 expression and the chemotactic responsiveness of DCs to CCL19 and CCL21, 17-19 the 2 CCR7 ligands known to promote DC entry into lymph nodes. [20][21][22] These observations opened the possibility that PGE 2 may also support the induction of antigen-specific immune responses by promoting the migration of Ag-carrying DCs to the draining lymph nodes and their interaction with lymph node-based naive and central memory T cells. Based on these observations, PGE 2 is frequently included in the cytokine cocktails 16 used to induce mature DCs for clinical use as vaccines against cancer. 23 Taking into account these apparently paradoxical effects of PGE 2 on DC functions and guided by the observations from...